Electrophysiological and biochemical effect of zinc oxide nanoparticles on heart functions of male Wistar rats

Zinc oxide nanoparticles (ZnO NPs) are one of the most abundantly used nanomaterials in cosmetics and topical products, and nowadays, they are explored in drug delivery and tissue engineering. Some recent data evidenced that they are responsible for cardiotoxic effects and systemic toxicity. The present study aimed to investigate the toxic effect of ZnO NPs (39 nm) on the heart of Wistar rats and to perform a dose–response relationship using three different dose levels (25, 50, 100 mg/kg bw) of ZnO NPs on the electrocardiogram (ECG) readings, the levels of biochemical function parameters of heart, and the oxidative stress and antioxidant biomarkers. Furthermore, zinc concentration level and histopathological examination of heart tissues were determined. ZnO NPs showed a dose-dependent effect, as the 100 mg/kg bw ZnO NPs treated group showed the most significant changes in ECGs parameters: R–R distance, P–R interval, R and T amplitudes, and increased levels of heart enzymes Creatine Kinase- MB (CK-MB) and Lactate dehydrogenase (LDH). On the other hand, elevated zinc concentration levels, oxidative stress biomarkers MDA and NO, and decreased GSH levels were found also in a dose-dependent manner, the results were supported by impairment in the histopathological structure of heart tissues. While the dose of 100 mg/kg bw of ZnO bulk group showed no significant effects on heart function. The present study concluded that ZnO NPs could induce cardiac dysfunctions and pathological lesions mainly in the high dose.

behavior of the rats were carefully recorded to observe the possible appearance of clinical signs following the treatment.The rats were divided into five equal groups (n = 6/group), and each group received a different treatment.The rats in each group were given i.p. injections every other day for ten days with the following: Control group: 0.5 ml of 0.9% normal saline as a vehicle; (2) Bulk group: ZnO bulk suspensions at a dose of 100 mg/kg bw; T1, T2, and T3 (ZnO NP-treated) groups: ZnO NPs suspension at doses of 25, 50 and 100 mg/kg bw, respectively.

Ethical approval
All of the animals used in the experiments received adequate care and the study protocol performed on animals complies with the guidelines of the National Institutes of Health and Ethical Conduct for Use and Care of Animals in Research Ethics Committee REC in Faculty of Medicine, Suez University (approval number: 19323).All methods were performed in accordance with the relevant guidelines (https:// arriv eguid elines.org).

Electrophysiological study
Anesthesia Rats' electrocardiogram recording was done in the five treated groups (one day before blood sampling).All treated rats were anesthetized with a dose of 1.25 g/kg bw urethane (Sigma-Aldrich, Germany), using i.p. injections, which is a widely used surgical anesthetic, since urethane can provide deep anesthetic effects while maintaining normal cardiovascular reflexes in the treated animals 28 .

ECG recordings
The ECG was recorded in the rats using a three-channel Digital ECG (ECG-300G, Biocare Bio-Medical co, Ltd, Germany) with a chart speed of 50 mm/second.The rats were placed in a dorsal position on a wooden platter, and four electrodes (Lead II) were attached to the skin of the forelimbs and hindlimbs 29,30 .Even though we used needle electrodes for the ECG recordings, the process was conducted in a quiet room to reduce stress.Gentle restrain and special care were used to obtain reliable readings from the rats.

Recorded ECG parameters
To assess the myocardial activity, we measured the heart rate (HR) and different ECG parameters such as conduction time (P-R interval), depolarization, and repolarization voltage (R and T waves) 31 .The HR was measured and calculated from the R-R interval.The changes were represented as chronotropic effects.The P-R interval (conduction time) was measured from the beginning of the P-wave to the start of the QRS complex, and the changes in the conduction times are called dromotropic effects.The amplitude of both R and T waves was measured from the isoelectric line up to the highest point of the waves 32 .

Sample collection and tissue preparation
After the end of the experiment, the rats were fasted overnight and weighed.Then, under the previous injection of urethane anesthesia blood samples were drawn from the retro-orbital venous plexus of the eyes using a capillary tube according to a previously described method 26 .Some of the blood samples were allowed to sit for 1 h at room temperature and then centrifuged at 2000×g for 3 min to collect the serum, which was then stored at − 22 °C for further biochemical assays.Finally, the animals were euthanized, and their hearts were excised and divided into two halves.The first half was fixed instantly in 10% neutral-buffered formalin for 2 days and used for histopathological examinations, whereas the other half was frozen for bioassays (− 80 °C).The frozen tissues were cut into small pieces and homogenized using 10 ml of 100 mM cold potassium phosphate buffer with 1 mM EDTA (pH 7.4) as previously described 27 .Then, the homogenate was centrifuged at 12,000×g for 30 min at 4 ℃, and the supernatant was removed to measure oxidative stress, antioxidant parameters, and zinc content.

Heart function biomarkers
Cardiac enzymes such as creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) were determined by enzymatic colorimetric method using commercial kits (SPINREACT Co., S.A.U, Spain).

Heart oxidative stress and antioxidant parameters
Determination of tissue-reduced glutathione (GSH).GSH was measured in the heart tissue of rats using a commercially available kit (Biodiagnostic, Egypt) as described previously 33 .
Determination of tissue nitric oxide (NO).NO was determined in the heart tissues of rats using a colorimetric kit (Bio diagnostic, Egypt) as described previously 34 .
Determination of tissue malondialdehyde (MDA).MDA was determined in the heart tissues of rats calorimetrically using a commercial kit (Biodiagnostic, Egypt) according to a previous method 35 .

Zinc ion concentration in heart tissues
The concentration of zinc ions (Zn ++ ) in each heart tissue of rats was measured using a Perkin Elmer-A Analyst 100 spectrophotometer using a previously discussed method 36 .

Ethics declarations
In accordance with ethical standards and guidelines for the use of animals in research, the animal experiments conducted in this study were approved by Research Ethics Committee REC in Faculty of Medicine, Suez University (approval number: 19323).All procedures involving animals were performed in compliance with relevant laws and regulations, and every effort was made to minimize any potential discomfort or distress to the animals.Prior to the commencement of the study, informed consent was obtained from the respective authorities, and proper measures were taken to ensure the humane and ethical treatment of the animals throughout the duration of the experiments.The details of the ethical approval and procedures are outlined in the "Methods" section of this manuscript, as required by the journal's submission guidelines (https:// arrive guidelines.org).

Consent to participate
All authors agree to participate in this study.

Experimental electrophysiology of control and treated groups
The ECG readings of rats (Table 1) showed that the T3 group (100 mg/kg BW) showed a significant decrease in HR (by 15.3%), whereas the T2 and T1 rats (50 and 25 mg/kg BW) showed nonsignificant decreases in HR (by 6.1% and 4%, respectively) compared to the control group.These changes in HR indicated a dose-dependent increase in ZnO NP toxicity (Fig. 1a).Conversely, the bulk group rats showed a nonsignificant decrease in HR (by 1.5%) compared to the control.Comparison between ZnO NPs and the bulk groups showed that the T3 group showed a significant decrease in HR (− 13.9%), whereas T2 and T1 showed nonsignificant decreases (− 4.6% and − 2.4%, respectively).Furthermore, the ECG results confirmed that the application of ZnO NPs induced a highly significant increase in the P-R intervals in the T3 group (by 39%), whereas the increases in the T2 and T1 groups were significant (by 21.95% and 19.5%, respectively) compared to the control group.This reveals the dose-dependent effect of the ZnO NPs (Fig. 1b).The ECGs of the rats in the bulk group displayed a significantly higher P-R interval (by 17%) than in the control group.Moreover, when we compared the effects in the ZnO-NP-treated and bulk groups, we observed a significantly higher P-R interval in the T3 group (by 18.75%), whereas the increases in the T2 and T1 groups were nonsignificant (by 4.16% and 2.08%, respectively).
The ECG data also showed that treatment with ZnO NPs caused a dose-dependent increase in the R-amplitude of rats' ECGs in the T1, T2, and T3 groups (Fig. 1c).It was significantly higher in all the groups (37.15%, 28.5% and 24.6% in T3, T2, and T1, respectively) compared to the control group.Furthermore, the bulk group showed a significant increase in R amplitude (21.78%) compared to the control.Comparison between the ZnO NP-treated and bulk groups revealed a nonsignificant increase in R amplitude in the T3, T2, and T1 (by 10%, 3.13%, and 2.14%, respectively).
The results also indicated a dose-dependent yet nonsignificant increase in T-amplitude in the ZnO NP-treated groups (by 9.7%, 3.1%, and 0.8% in the T3, T2, and T1 groups, respectively) compared to the control group (Fig. 1d).Moreover, the bulk group showed a nonsignificant increase by 0.4% compared to the control group.Compared to the bulk group, the ZnO NP-treated groups showed nonsignificant increases in T-amplitude (by 9.2%, 2.7%, and 0.38% in the T3, T2, and T1 groups, respectively).
From the changes observed in the ECG parameters between control and ZnO NP-treated groups, such as R and T amplitudes (Plate 1), we deduced that the rats in the T3 group exhibited cardiac disorders as represented by the increased R-R distance (bradycardia), longer P-R intervals and missing P waves (first-degree block) (Plate 2).

Heart function enzymes
The biochemical data on heart function biomarkers (Table 2) demonstrated that the serum levels of CK-MB in the ZnO NP-treated rats showed an extremely significant increase (by 288.8%) in the T3 group, whereas that in the T2 group showed a highly significant increase (by 55.7%).Conversely, in the T1 group, there was a nonsignificant decrease (by − 2.68%) compared to the control group.This depicted the dose-dependent response in CK-MB levels in the three ZnO NP-treated groups (Fig. 2a).The data also revealed that ZnO bulk-treated rats produced a nonsignificant decrease in CK-MB levels by − 8% compared to the control group.Compared to the bulk group, the data showed a highly significant increase in CK-MB in both T3 and T2 groups by 322.7% and 69.3%, respectively, whereas the T1 rats demonstrated a nonsignificant increase of 5.8%.
The data also showed that serum LDH levels were significantly increased in the T3 group by 35.2%.However, the increase in the T2 group was nonsignificant (by 9.66%), whereas the T1 group showed a nonsignificant decrease (by − 1.8%) compared to the control.This clarifies the dose-dependent effect of ZnO NPs (Fig. 2b).While bulk ZnO-treated rats produced a nonsignificant increase (by 1.2%) compared to the control.Compared to the bulk group, the LDH levels were significantly higher in the T3 group (33.6%).Furthermore, the T2 group showed a nonsignificant increase in LDH (8.35%), whereas T1 showed a nonsignificant decrease (− 2.97%).www.nature.com/scientificreports/

Oxidative stress and antioxidant biomarkers levels in the heart tissues
The data on the oxidative stress and antioxidant biomarkers levels demonstrated that ZnO NP-treated rats showed a significant decrease in reduced glutathione (GSH) levels in rats in T3 and T2 (− 40.77% and − 26.88%, respectively) compared to the control group (Table 3).Conversely, the rats in the T1 group showed a nonsignificant decrease in GSH levels (− 11.6%).This indicated the dose-dependent effect of ZnO NPs (Fig. 3a).However, the bulk group showed a nonsignificant increase in the GSH levels (2%) compared to the control.When we compared the GSH levels in the ZnO NP-treated and the bulk groups, the GSH levels in the T3 and T2 groups were significantly lower (by − 42.16% and − 28.6%, respectively), whereas T1 showed a nonsignificant decrease (− 13.5%).
The data also confirmed the dose-dependent effect of ZnO NPs on the nitric oxide (NO) levels in the heart tissues of the treated rats (Fig. 3b).The NO levels showed a highly significant increase in the T3 group (102%) and a significant increase in the T2 group (53.9%).Whereas the T1 group showed a nonsignificant increase (1.2%) compared to the control.However, the NO levels in heart tissues of the bulk group showed a nonsignificant     decrease (− 10.5%) compared to the control.Comparison of the NO levels in the ZnO NP-treated and the bulk groups revealed a highly significant increase in the T3 and T2 groups (111.2% and 63.2%, respectively), whereas the T1 group showed a nonsignificant increase (11.5%) compared to the bulk group.We observed that the malondialdehyde (MDA) levels in the heart tissues of the ZnO NP-treated rats were highly significantly increased in the T3 group (69%), significantly increased in T2 (37.9%), and nonsignificantly increased in T1 groups (10.7%), proving the dose-dependent effect of ZnO NPs (Fig. 3c) However, the bulk group showed a nonsignificant decrease in MDA levels (− 2.25%) compared to the control group.Furthermore, compared to the bulk group, the T3, T2, and T1 groups showed highly significant, significant, and nonsignificant increases in MDA levels (by 68.85%.38.9%, and 12.5%, respectively).

Zinc ion concentration in the heart
We assessed the zinc ions (Zn ++ ) concentrations in the heart tissues of rats (Table 4).The increases in the Zn ++ content in the T3, T2, and T1 groups were highly significant (103.6%),significant (31.55%), and nonsignificant (6.1%), respectively.Again, this indicated the dose-dependent effect of ZnO NPs on the zinc ions concentrations (Fig. 4).The bulk group showed a nonsignificant increase in Zn ++ content by 4.55% compared to the control group.Compared to the bulk group, the increases in Zn concentration were nonsignificant in T1 (1.5%), significant in the T2 group (25.8%), and highly significant in T3 (94.7%).

Histopathological observation of the heart
The histopathological observations in heart sections of the control rats showed normal (arrowheads) and regularly arranged cardiac myofibres (red arrows).Intercalated disks (blue arrows) were also seen (Fig. 5a).However, the cardiac sections of the T1 group showed vascular congestion (asterisks) and the nearby muscles were darker and degenerated (black arrows) (Fig. 5b).The heart sections of the T2 rats showed marked vascular congestion (asterisks), thickening of vessel walls along with darkening and degeneration (black arrows) of the nearby muscles.We also observed mild myocyte degeneration (black arrows) (Fig. 5c).Similarly, the heart sections of the T3 group showed marked vascular congestion (asterisks), and the nearby muscles were darker and degenerated (black arrows).Interestingly, we found myofibre dislocation in the cardiac muscle (red asterisks) in this group (Fig. 5d).Whereas the heart sections of the bulk group showed normal cardiomyocytes (arrowheads), regularly arranged cardiac myofibres (red arrows), and intercalated disks (blue arrows) (Fig. 5e).

Discussion
One of the most important safety considerations in preclinical and clinical investigations is evaluating the cardiotoxicity of novel chemicals and drugs 38 .Physiologically, it has been established that increased plasma levels of CK are linked to skeletal or cardiac muscle damage, whereas increased serum LDH levels indicate myocardial infarction 22,39 .CK-MB and LDH parameters are accepted for the clinical diagnosis of myocardial infarction by the WHO 40 .
The biochemical data on the CK-MB and LDH levels in our study suggested that treatment with ZnO NPs increased the levels of both enzymes in a dose-dependent manner.The most significant change indicated by the highest percentage increase was implicated with the highest dose (100 mg/kg BW).An earlier study reported that oral injections with ZnO NPs at doses of 100 and 400 mg/kg BW for 28 days caused a significant increase in the serum cardiac markers levels, as well as alteration in the cardiac tissues associated with elevation in oxidative stress biomarkers 41 .Additionally, the accumulation of nanoparticles in the heart tissues was associated with increased CK and LDH levels in the blood and has been attributed to the disruption in the plasma membrane structure and increased permeability 42 .www.nature.com/scientificreports/Since electrical activity is a distinctive characteristic of the heart, any interruption in this activity or the contractile properties reflects pathogenesis 43 .ECG recordings provide a standardized method to evaluate the pharmacological and toxicological effects of drugs and chemicals on the heart 44 .
In this study, the ECG recordings of the anesthetized rats demonstrated the toxic effects of ZnO NPs on the myocardium as shown by the negative chronotropic effects (bradycardia) represented by a significant decrease in HR induced due to ZnO NP injection.We also observed a negative dromotropic effect represented by the significant prolongation in P-R, which reflects the delay in conduction velocity and disturbance in the electrical conductivity in the atria and the AV node.These effects occurred in a dose-response manner, and the most pronounced effects were seen at the highest dose (100 mg/kg bw).
We also proved that the application of ZnO NPs significantly increased the ventricular depolarization voltage demonstrated by the increased R-wave amplitude.The dose-dependent nature of this positive inotropic effect was demonstrated by the increase in contractile force of the ventricle with an increase in dosage.The dose-dependent increase in the ventricular repolarization voltage (T voltage) was also seen in the three ZnO NP-treated groups (T3, T2, and T1) although it was statistically nonsignificant compared to the control group.
Treatment with bulk ZnO did not affect the HR and T voltages in the rats as they were similar to the control rats.Although a significant increase in the P-R interval and R voltage was observed in this group, the positive inotropic effect and the ventricular contractile force were less than that seen in the ZnO NP-treated groups, which confirmed that the bulk form is safer than the NPs in terms of cardiotoxicity.
The present electrophysiological manifestations of ZnONPs, mainly at the highest dose, demonstrated an important point that the negative chronotropic and dromotropic effects are accompanied by positive inotropic effects.Moreover, they showed the presence of cardiac disorders, the most common being bradycardia and 1stdegree atrioventricular block.
Our data was consistent with a study performed on zebrafish embryos which showed significant bradycardia after being exposed to silica nanoparticles.These effects developed with the increase in nanomaterial concentration 45 .Moreover, ZnO NPs were reported to decrease HR and cell contractility in human cardiomyocytes 4 .
The AV node and His-bundle typically form the only electrically active connection between atria and ventricles.Therefore, the presence of bradycardia and P-R interval elongation indicates a possible lesion in the sinoatrial (SA), atrioventricular (AV) nodes, and atrioventricular conduction 46 .Our findings showing the decrease in the HR along with the progressive increase in R-wave amplitude and P-R interval elongation were consistent with the results of a previous study, which reported that catecholamines-induced cardiotoxicity indicated the toxic effects on atrioventricular (AV) and sinoatrial (SA) nodes, destruction of myocytes, and/ or myocardial infarction 47 .
The majority of myocardial alterations can be attributed to increased oxidative stress.Furthermore, the increased production of myocardial oxidants has been suggested as the main cause of heart failure.The malfunction of the Na+/K+ ATPase pump and myocardium-imbalanced electrolyte levels has also been implicated 48 .Consistent with these findings, our data revealed that ZnO NP-induced cardiotoxicity is associated with oxidative stress development and impairment in antioxidant balance, which occurred in a dose-dependent manner as the highest dose group (T3) showed a highly significant increase in heart NO and lipid peroxidation (MDA) levels.Furthermore, there was a significant depletion in heart GSH levels.Meanwhile, treatment with ZnO bulk did not show any significant changes in either heart NO or lipid peroxidation levels in the heart tissues of rats.Additionally, no significant change in the GSH levels was seen, confirming its safety.These results also supported our previous findings on the hepatotoxic effect of ZnO NPs in rats where we showed increased oxidative stress production and depletion in antioxidant levels in both serum and tissues 14 .
ROS plays a crucial role in the impairment of the cardiomyocyte microenvironment due to exposure to NPs, which was proved by increased ROS production, altered redox homeostasis, and diminished antioxidant ability, resulting in damage to the cardiac muscles 8 .The overproduction of NO levels in tissues can lead to severe cellular damage.NO may cause constriction of coronary arteries and contribute to myocardial ischemia in patients with coronary artery disease, also NO could be relevant to oxidative stress and or nitrosative stress processes due to its chemical nature when it is excessively produced it could participate in the formation of biologically reactive nitrogen oxide species (RNOS) that are mediated in oxidative nitration and oxidative damage of biomolecules such as lipids, proteins, and DNA 13 .
Zinc is an essential nutrient for human health and is vital for the physiological functions of various organs, owing to its important role in enzymes and proteins 49 .ZnO NPs were reported to induce more toxic effects than larger (bulk) ZnO particles due to their larger surface-to-mass ratio, which enhanced their solubility in biological systems 50 .Heavy metals enhance toxicity when they cannot be metabolized in the body and are accumulated in soft tissues 51 .Our findings showing the estimated zinc concentrations in heart tissues shed light on the mechanism of ZnO NPs cardiotoxicity.
The results showed that injection with the highest dose of ZnO NPs induced a highly significant increase in zinc ion (Zn++) concentrations in the heart tissues, whereas the bulk-treated group showed a nonsignificant increase in heart Zn++ concentrations compared to the control.As discussed previously, the accumulation of Zn++ in the hepatic tissues due to the administration of the same dose of ZnO NPs was associated with other biochemical and histological indices of the liver of rats 14 .
Recent studies revealed that increased production of Zn2+ during the excitation-contraction cycle in the cardiomyocytes indicated oxidative stress, which could trigger further production of pro-oxidants leading to oxidative damage in cardiomyocytes and heart dysfunction 52 .
The histopathological assessment of the rats' heart sections showed dose-dependent alterations in the cardiac tissues of rats as the cardiac sections of the T3 group (highest dose) exhibited highly marked vascular congestion and degeneration along with myofibre dislocation, confirming the biochemical and electrophysiological effects of the ZnO NPs.While the T1 and T2 groups showed mild degeneration and congestion, the bulk group exhibited normal cardiomyocytes, supporting our biochemical findings.Consistent with our results, a report showed that oral administration of 15 nm-sized ZnO NPs in rats caused significant pathological effects in the heart, including degeneration of the heart muscles, focal hemorrhages, and bleeding along with severe anemia 53 .

Conclusion
In conclusion, this study demonstrated that the subacute i.p. injection of ZnO NPs induced cardiac dysfunctions and pathological lesions in a dose-dependent manner, whereas the ZnO bulk form did not exert any toxicity.Our data also suggest that the accumulation of Zn++ in the heart tissues, accompanied by increased oxidative stress and depletion in the antioxidant activity can be the possible underlying mechanisms behind ZnO NPs' toxic effects on both the mechanical and electrical activities of the cardiomyocytes.However, further in-depth investigation is needed at the molecular level to fully understand the mechanism of ZnO NPs' cardiotoxicity.

Table 2 .
Levels of heart enzymes, CK-MB, and LDH in control, bulk, and three ZnO NP-treated groups.CK-MB creatine kinase-MB, LDH lactate dehydrogenase, ZnO NP-treated groups T1: 25 mg/kg BW, T2: 50 mg/kg BW, T3: 100 mg/kg BW, bulk ZnO bulk treated group (100 mg/kg BW), SE standard error.*p < 0.05, **p < 0.001, ***p < 0.0001 compared to control group.Values are shown as mean ± SE (n = 6) and the superscript in different letters indicates the following: a significant compared to the respective control group.b Significant compared to the bulk group.A: percentage of change as compared to the control group.B: percentage of change as compared to the bulk group.

Figure 3 .
Figure 3. Represents the dose-response relationship in heart oxidative stress and antioxidant parameters following the treatment of ZnO NPs at three different doses.ZnO NPs: zinc oxide nanoparticles; ZnO NPs three doses: 25 mg/kgbw, 50 mg/kgbw, and 100 mg/kgbw; (a) GSH: heart reduced glutathione, (b) NO: heart nitric oxide, and (c) MDA: heart malondialdehyde.% Percentage of change as compared to the control group.**p < 0.001,***p < 0.0001 compared to control group.

Table 4 .
Zinc ion concentrations in heart tissues in control and treated groups.Zn ++ zinc ions, conc.Concentration, ZnO NP-treated groupsT1: 25 mg/kg BW, T2: 50 mg/kg BW, T3: 100 mg/kg BW, bulk ZnO bulk treated group (100 mg/kg bw), SE standard error.*p < 0.05, **p < 0.001, ***p < 0.0001 compared to the control group.Values are shown as means ± SE (n = 6) and the superscripts in different letters indicate the following: a significant compared to the respective control group.b Significant compared to the bulk group.A: percentage of change as compared to control group.B: percentage of change as compared to the bulk group.

Figure 4 .
Figure 4. Represents the dose-response relationship in heart zinc ions concentration following the treatment of ZnO NPs at three different doses.ZnO NPs: zinc oxide nanoparticles; ZnO NPs three doses: 25 mg/kgbw, 50 mg/kgbw, and 100 mg/kgbw; conc.concentration.% Percentage of change as compared to the control group.**p < 0.001,***p < 0.0001 compared to control group.

Figure 5 .
Figure 5.Effect of ZnO NPs and bulk form on histopathology of heart tissues of treated rats.Heart sections stained with H&E (×400), Scale bar represent 25μm (a) Heart tissues of control rats showing the normal appearance of cardiac myofibers, single oval and centrally located nuclei of cardiomyocytes (arrowheads), regularly arranged cardiac myofibres (red arrows), and intercalated disks (blue arrows), (b) heart tissues of ZnO NPs T1 (25 mg/kgbw) treated rats showing vascular congestion (black asterisk) nearby muscles looked darker and degenerated (black arrows), (c) heart tissues of ZnO NPs T2(50 mg/kgbw) treated rats showed marked vascular congestion (black asterisks), moreover, thickening of vessel walls.The nearby muscles looked darker and degenerated (black arrows), and mild myocyte degeneration was found (black arrows), (d) heart tissues of ZnO NPs T3 (100 mg/kgbw) treated rats showed marked vascular congestion (black asterisks) nearby muscles looked darker and degenerated (black arrows), and also dislocation of myofibres (red asterisks), (e) heart tissues of ZnO bulk (100 mg/kgbw) treated rats showed the normal structure of cardiomyocytes.Oval and centrally located nuclei of cardiomyocytes (arrowheads), regularly arranged cardiac myofibres (red arrows), and intercalated disks (blue arrows).ZnO NPs zinc oxide nanoparticles, H&E hematoxylin and eosin.

Table 1 .
Electrocardiogram (ECG) parameters of control, bulk, and the three ZnO NP-treated groups (T1, T2, and T3).A: percentage of change compared to the control group.B: percentage of change compared to the bulk group.HR heart rate, PR interval conduction time, R-amplitude depolarization voltage, T-amplitude repolarization voltage, ZnO NPs zinc oxide nanoparticles, ZnO NP-treated groups T1: 25 mg/kg BW, T2: 50 mg/kg BW, T3: 100 mg/kg BW, Bulk ZnO bulk treated group (100 mg/kg BW), SE standard error.*p < 0.05, **p < 0.001, ***p < 0.0001 compared to the control group.Values are shown as mean ± SE (n = 6), and the superscripts in different letters represent the following: a significant compared to the respective control group, b significant compared to the bulk group.